disrupting e. coli cells

In article <2lo72q$9ur at news.iastate.edu>, bipin at iastate.edu (Bipin K Dalmia) writes:
|> i've never had much success with sonication but since that is the only
|> piece of equipment we have at present, do you have any suggestions for
|> optimizing e. coli disruption with a sonicator? i've heard that some
|> people use lysozyme treatment before sonicating, do you have any
|> experience with this?? any other treatments??
|>|> bip
|> --
|> bipin k. dalmia the other night i was lying on my bed, looking
|>bipin at iastate.edu up at the beautiful stars, and i said to myself,
|>n2.bkd at isumvs.iastate.edu 'where the F*CK is my ROOF !!'
|> --
I've used sonication, with and without lysozyme, and also freeze-thawing
to lyse coli
(freeze-thawing as in multiple cycles of flash freezing in liquid N2,
then rapid thawing in a warm water bath (we use 65C, but do't let the samples get
to 65, or much above 30 really, and only invert gently)
I've had a _lot_ more sucess with sonication than with freeze- thawing,
and found that adding lysozyme wasn't really worth it (you're contaminating your
prep with another protein, to start with).
A couple of good things to do when you're sonicating are
tune the probe first (Does make a difference)
do everything on ice, and let it cool down for 10min between
cycles
resuspend the cell debris after centrifugation, and resonicate it
(basically the same as if it was fresh cells, just maybe a little more gently) -
can get another 20% yield easy wdoing this.
The only other peice of advice I can think of is to keep sonicating,
spinning and sonicating, and check by gel to see whether the cell debris still
contains intact cells. Should be able to tell farily easily.
Good luck (cells that won't lyse are a pain - I sympathise)
Ben
--
______________________________________________________________________________
Ben Davis,
MRC Protein Function and Design,
Cambridge, UK
______________________________________________________________________________
"They can make me do it, but they can't make me do it with dignity."